High-Speed, Comprehensive, Two Dimensional Separations of Peptides and Small Molecule Biological Amines Using Capillary Electrophoresis Coupled with Micro Free Flow Electrophoresis

Alexander C. Johnson, Michael T. Bowser

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

Abstract

Two-dimensional (2D) separations are able to generate significantly higher peak capacities than their one-dimensional counterparts. Unfortunately, current hyphenated 2D separations are limited by the speed of the second dimension separation and the consequent loss of peak capacity due to under sampling of peaks as they elute from the first dimension separation. Continuous micro free flow electrophoresis (μFFE) separations eliminate under sampling as a limitation when incorporated as the second dimension of a 2D separation. In the current manuscript we describe the first coupling of capillary electrophoresis (CE) with μFFE to perform 2D CE × μFFE separations. The CE separation capillary was directly inserted into the μFFE separation channel using an edge on interface. Analyte peaks streamed directly into the μFFE separation channel as they migrated off the CE capillary. No complicated injection, valving, or voltage changes were necessary to couple the two separation modes. 2D CE × μFFE generated an ideal peak capacity of 2 592 in a 9 min separation of fluorescently labeled peptides (7.6 min separation window, 342 peaks/min). Data points were recorded every 250-500 ms (>8 data points/peak), effectively eliminating under sampling as a source of band broadening. CE × μFFE generated an ideal peak capacity of 1885 in a 2.7 min separation of fluorescently labeled small molecule bioamines (1.8 min separation window, 1053 peaks/min). Peaks in the 2D CE × μFFE separation of peptides covered 30% of the available separation space, resulting in a corrected peak capacity of 778 (102 peaks/min). The fractional coverage of the 2D CE × μFFE separation of small molecule bioamines was 20%, resulting in a corrected peak capacity of 377 (209 peaks/min).

Original languageEnglish (US)
Pages (from-to)1665-1673
Number of pages9
JournalAnalytical Chemistry
Volume89
Issue number3
DOIs
StatePublished - Feb 7 2017

Bibliographical note

Funding Information:
This research was funded by the National Science Foundation (Grant CHE-1152022) and the National Institutes of Health (Grant GM063533)

Publisher Copyright:
© 2016 American Chemical Society.

Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.

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